Motility contrast imaging is a coherence-domain imaging technique that uses wide-field illumination and is multimodal in operation, particularly as the technique allows the simultaneous illumination of a large number of diffraction-limited areas. Unlike imaging operations performed in accordance with single-mode optical coherence tomography (OCT), motility contrast imaging allows internal motion of living tissue to be extracted as a function of three-dimensional location.
Motility contrast imaging is an extension of holographic optical coherence imaging (HOCI). Optical coherence imaging (OCI) was originally developed by French and Nolte using adaptive optical films. OCI images were taken of living tissue, and later enhancements significantly improved the sensitivity and penetration depth. OCI included speckle-reduction elements (vibrating mirrors and diffusers) with time-averaging to remove speckle to allow imaging of structures. However, it was also discovered that the highly active dynamic speckle in living tissue vanished in dead or chemically-fixed tissue.
The speckle-reduction elements were removed to study this dynamic speckle in living tissue directly. This change in emphasis prompted a migration of the detection technology to conventional CCD cameras using digital holography, particularly to detect sub-cellular motion inside tissue with high sensitivity. Digital holography is less complex and requires no sophisticated nonlinear optical elements.
Motility contrast imaging (MCI) uses fully-developed speckle fields that carry little structural information. More particularly, MCI identifies the separate contributions of different types of sub-cellular motion to measured dynamic speckle, which enables the development of an endogenous multi-functional imaging method based on the different signatures from the different functions of intracellular motion. To provide these fully-developed speckle fields, multimode illumination by low-coherence light is used. This is in sharp contrast to OCT, which seeks to eliminate speckle to achieve the highest possible spatial resolution. The speckle-fields of MCI arise from the interference of multiple scatterers with random phases within a coherence volume inside the tissue. The holographic coherence gate localizes the detected motion to within a thin slab inside the tissue with a thickness determined by the coherence length of the laser. Using this approach, nanoscale motion has been sensed as deep as 1 mm inside tissue localized to within 30 micron volumes (voxel size corresponding to the spatial resolution) across a field of view of 1 mm. MCI presents an unexpected imaging approach based on motility as the contrast agent.
One of the many possible applications of MCI is to image the effects of the largest class of anti-cancer drugs—i.e., the anti-mitotic drugs that arrest cellular motion associated with mitosis. The many different forms of intracellular motion contribute separately to the measured dynamic speckle. With the present invention it is possible to unravel these different contributions by using selected drugs that arrest different types of motion. To test these drugs, multicellular tumor spheroids are used, which are three-dimensional tissue structures that reproduce in vivo pharmacological responses with all the advantages of being in vitro. Many possible tissue targets can be investigated with this invention, including three-dimensional tissue models of specific cancers as well as connective tissue models that closely mimic the extracellular matrix.
According to a first aspect of the present invention, a system for motility contrast imaging a biological target within tissue is provided. The system comprises a CCD array located at a Fourier plane; an illumination source for generating an incoming beam; a first beam splitter for receiving the incoming beam and producing an object beam and a reference beam; a second beam splitter for causing the object beam to illuminate the biological target and for directing a backscattered object beam towards the CCD array; a computer-controlled delay stage for zero-path-matching the reference beam to the backscattered object beam; a reference beam intersects the backscattered object beam at an angle to produce a series of interference fringes that modulate Fourier-domain information; and a computer for receiving a time sequence of Fourier-domain information. The interference fringes between the backscattered object beam and the reference beam are recorded by the CCD array and passed to the computer which constructs a digital hologram at successive times.
According to another aspect of the present invention, a system for motility contrast imaging of multiple biological targets is provided. The system comprises a multi-well plate having a plurality of wells, each well containing a biological target; a CCD array located at a Fourier plane; an illumination source for generating incoming beams; a first beam splitter for receiving the incoming beams and producing a multitude of object beams that illuminate the biological target; a microlens array for directing a backscattered object beam towards the CCD array; a computer-controlled delay stage for zero-path-matching the reference beam to the backscattered object beam; a reference beam that intersects the backscattered object beam at an angle to produce a series of interference fringes that modulate Fourier-domain information; and a computer for receiving the Fourier-domain information. The interference fringes between the backscattered object beam and the reference beam are recorded by the CCD array and passed to the computer which constructs a digital hologram of the first portion of the plurality of wells that contain biological targets.
In accordance with yet another aspect of the present invention, a method for motility contrast imaging biological targets within tissues is provided. The method comprises generating an incoming beam with an illumination source; receiving the incoming beam with a first polarizing beam splitter to produce an object beam a reference beam; causing the object beam to illuminate the biological target; directing a backscattered object beam towards the CCD array; zero-path-matching the reference beam to the backscattered object beam; intersecting the reference beam with the backscattered object beam at an angle to produce a series of interference fringes that modulate Fourier-domain information; recording the interference fringes between the backscattered object beam and the reference beam; and using the Fourier-domain information to construct a digital hologram of a first portion of a plurality of wells containing the biological targets.